Team:UConn/Results
Results
In this iGEM project, we set out to explore the world of algae and biofuel production. There have only been a few projects in iGEM relating to algae, so we aimed to expand the parts that could be used for algal genetic engineering.
One of the important components of expression in algae and other eukaryotic cells is using a promoter and terminator system that will be highly transcribed in the cell. Through literature research, we identified a promoter and terminator (VCP2 and VCP1) that we could convert to biobricks. These were synthesized by IDT with biobrick regions added and were cloned into a standard backbone. The expected length of both the VCP1 and VCP2 sequences in standard backbones were 2.8 kb, and we were successful in ligating the VCP1 and VCP2 sequences to the standard backbone, as we were able to obtain gels with bands that matched their expected lengths, as shown in the figure below:
After successfully cloning the VCP1 promoter and VCP2 terminator into the standard backbone, we had to test to see if they were effective in promoting DNA expression in Nannochloropsis oceanica. We decided the best way to go about this was by creating a VCP1-GFP-VCP2 construct and attempting to get the algae to express the construct.
First, a VCP1-GFP construct was assembled through digesting the GFP out of its original backbone and then ligating into the backbone containing the VCP1. This process was successful, as shown by the gel shown on the left. We confirmed the correct length of this part by comparing it to a virtual digest, shown above the actual gel.
Next, VCP2 was digested out of its plasmid backbone and ligated into the VCP1-GFP construct, to create a VCP1-GFP-VCP2 construct, which we referred to as the VGV complex. In order to determine that this construct was correctly ligated, we ran a gel and compared it to a virtual digest. After examining the results, we determined that the VGV was the correct length, indicating it was correctly ligated.
This VGV complex (shown here on the right) was then prepared for electroporation through numerous transformations and plasmid minipreps in order to increase the amount of VGV available. Several attempts of electroporation were performed, however the effectiveness of the electroporation was not able to be determined due to time constraints.
In order to determine the effectiveness of electroporation, one needs to culture the resultant algae in order to properly evaluate transformation efficiency. Additionally, transformation attempts need a selection marker to select for algae expressing the construct. Hygromyocin was chosen as our selection marker. We successfully edited and cloned a Hygromyocin B resistance gene into a standard backbone for future use.
Our goal was to increase the fatty acid content of the algae and introduce a secretion method of fatty acids to allow harvesting of lipids to be used in biofuel. Increasing the fatty acid content of the algae requires manipulation of the enzymes involved in fatty acid production and storage. The mRNA transcripts of Nannochloropsis oceanica were analyzed for these proteins, and a couple of them were targeted for PCR out of a DNA extraction.
There were four different mRNA transcripts we targeted in particular, and they were indentified as mRNA 5911 (2.1kb), mRNA 10796 (1.3kb), mRNA 6531 (2.3kb), mRNA 5940 (2.2kb). We were somewhat successful in isolating these sequences from the DNA extracted. We investigated the results of the PCR reactions through gel electrophoresis, the results of which are shown on the right. The only mRNA sequence that was not successful in being isolated was mRNA 5940. In the gel images, bands that are circled are samples we deemed as correct.
As for the fatty acid excretion method, we identified an ATP-Binding Cassette (ABC transporter) present in the plant Arabidopsis thaliana that functions to excrete fatty acids onto its surface to give it a waxy exterior. Since algae are closely related to plants, we determined that this gene may work transgenically, and inserting the gene into Nannochloropsis oceanica may give the algae a method of excreting fatty acids.
Instead of extracting the gene for the ABC transporter from Arabidopsis thaliana, we decided to synthesize the gene by using an IDT order. We attempted to clone this transporter into the standard backbone, however this cloning was not successful. This result was not surprising, as the part was not synthesized up to standard, so the chance of it working was very low.
Given more time, there was a lot more we would have liked to do with this project. Electroporation of the algae culture could test the effectiveness of the VGV construct. Since GFP was cloned into the region, GFP expression in the algae could determine the effectiveness of the system.
Additionally, the other constructs we investigated would be tested. These constructs include the Hygromyocin resistance genes, the ABC transporter, and the mRNA sequences we isolated from the Nannochloropsis oceanica genome. These sequences would be placed in between the VCP1 promoter and VCP2 terminator to create a plasmid that the algae would read and express, improving its lipid production and excretion.
A plan for a continuous photosynthetic culturing system was in the initial stages of design and development. This system would entail integrating an algae culture into a mesh surrounding a bucky-ball type system. This ball would then be rotated continuously while submerged in media to ensure that the algae were receiving equal light and nutrients. The algae in the mesh would have the gene for the ABC transporter, and the lipids would be excreted into solution with the media, where they would then be separated out after draining the media from the container. Preliminary sketches and CAD designs of this system are shown to the right.
Algae Culturing
We set up an algae culture and maintained this culture over the course of the project, to be used for DNA extraction and possibly electroporation in later steps. The algae were set up using various dilutions of media to algae culture. The media used in all cultures was F/2 media (without silicate). As the project went on, we continuously split the cultures, and went from just a few tubes of startup culture at the beginning to almost twenty dense, happy, and healthy cultures by the end!Improving Algae DNA Expression
One of the important components of expression in algae and other eukaryotic cells is using a promoter and terminator system that will be highly transcribed in the cell. Through literature research, we identified a promoter and terminator (VCP2 and VCP1) that we could convert to biobricks. These were synthesized by IDT with biobrick regions added and were cloned into a standard backbone. The expected length of both the VCP1 and VCP2 sequences in standard backbones were 2.8 kb, and we were successful in ligating the VCP1 and VCP2 sequences to the standard backbone, as we were able to obtain gels with bands that matched their expected lengths, as shown in the figure below:
Testing Effectiveness of DNA Expression
After successfully cloning the VCP1 promoter and VCP2 terminator into the standard backbone, we had to test to see if they were effective in promoting DNA expression in Nannochloropsis oceanica. We decided the best way to go about this was by creating a VCP1-GFP-VCP2 construct and attempting to get the algae to express the construct.
First, a VCP1-GFP construct was assembled through digesting the GFP out of its original backbone and then ligating into the backbone containing the VCP1. This process was successful, as shown by the gel shown on the left. We confirmed the correct length of this part by comparing it to a virtual digest, shown above the actual gel.
Next, VCP2 was digested out of its plasmid backbone and ligated into the VCP1-GFP construct, to create a VCP1-GFP-VCP2 construct, which we referred to as the VGV complex. In order to determine that this construct was correctly ligated, we ran a gel and compared it to a virtual digest. After examining the results, we determined that the VGV was the correct length, indicating it was correctly ligated. This VGV complex (shown here on the right) was then prepared for electroporation through numerous transformations and plasmid minipreps in order to increase the amount of VGV available. Several attempts of electroporation were performed, however the effectiveness of the electroporation was not able to be determined due to time constraints. In order to determine the effectiveness of electroporation, one needs to culture the resultant algae in order to properly evaluate transformation efficiency. Additionally, transformation attempts need a selection marker to select for algae expressing the construct. Hygromyocin was chosen as our selection marker. We successfully edited and cloned a Hygromyocin B resistance gene into a standard backbone for future use.
Increasing Fatty Acid Content and Excretion
Our goal was to increase the fatty acid content of the algae and introduce a secretion method of fatty acids to allow harvesting of lipids to be used in biofuel. Increasing the fatty acid content of the algae requires manipulation of the enzymes involved in fatty acid production and storage. The mRNA transcripts of Nannochloropsis oceanica were analyzed for these proteins, and a couple of them were targeted for PCR out of a DNA extraction.
There were four different mRNA transcripts we targeted in particular, and they were indentified as mRNA 5911 (2.1kb), mRNA 10796 (1.3kb), mRNA 6531 (2.3kb), mRNA 5940 (2.2kb). We were somewhat successful in isolating these sequences from the DNA extracted. We investigated the results of the PCR reactions through gel electrophoresis, the results of which are shown on the right. The only mRNA sequence that was not successful in being isolated was mRNA 5940. In the gel images, bands that are circled are samples we deemed as correct.
As for the fatty acid excretion method, we identified an ATP-Binding Cassette (ABC transporter) present in the plant Arabidopsis thaliana that functions to excrete fatty acids onto its surface to give it a waxy exterior. Since algae are closely related to plants, we determined that this gene may work transgenically, and inserting the gene into Nannochloropsis oceanica may give the algae a method of excreting fatty acids.
Instead of extracting the gene for the ABC transporter from Arabidopsis thaliana, we decided to synthesize the gene by using an IDT order. We attempted to clone this transporter into the standard backbone, however this cloning was not successful. This result was not surprising, as the part was not synthesized up to standard, so the chance of it working was very low.
Future Plans
Given more time, there was a lot more we would have liked to do with this project. Electroporation of the algae culture could test the effectiveness of the VGV construct. Since GFP was cloned into the region, GFP expression in the algae could determine the effectiveness of the system.
Additionally, the other constructs we investigated would be tested. These constructs include the Hygromyocin resistance genes, the ABC transporter, and the mRNA sequences we isolated from the Nannochloropsis oceanica genome. These sequences would be placed in between the VCP1 promoter and VCP2 terminator to create a plasmid that the algae would read and express, improving its lipid production and excretion.
A plan for a continuous photosynthetic culturing system was in the initial stages of design and development. This system would entail integrating an algae culture into a mesh surrounding a bucky-ball type system. This ball would then be rotated continuously while submerged in media to ensure that the algae were receiving equal light and nutrients. The algae in the mesh would have the gene for the ABC transporter, and the lipids would be excreted into solution with the media, where they would then be separated out after draining the media from the container. Preliminary sketches and CAD designs of this system are shown to the right.